KR20080014144A - Dosing schedule for erbb2 anticancer agents - Google Patents

Abstract

The invention is directed to methods for the a method for treating overexpression of the erbB2 in a mammal in need of treatment by administering to the mammal a therapeutically effective amount of a first inhibitor of an erbB2 receptor and then, after an interval of less than 24 hours, administering to the mammal from one to six therapeutically effective amounts of the same or different inhibitor of the erbB2 receptor. The invention is also directed to a slow daily infusion of the erbB2 inhibitor. The overexpression of the erbB2 receptor can result in abnormal cell growth and lead to cancer. By the methods of the invention, the efficacy and safety of the inhibitors is increased. The invention is also directed to kits for facilitating the dose administration method of the invention.

Description

ｅｒｂＢ２ Dosing schedule for erbB2 anticancer agents

FIG. 1 shows inhibitor E-2-methoxy-N- (3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) administered PO, QD to mice with FRE / erbB2 tumors. Antitumor efficacy of) -phenylamino) -quinazolin-6-yl} -allyl) -acetamide. The ordinate is a measure of tumor growth on day 7 compared to the vehicle control.

FIG. 2 shows inhibitor E-2-methoxy-N- (3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) administered IV, QD to mice with FRE / erbB2 tumors. Antitumor efficacy of) -phenylamino) -quinazolin-6-yl} -allyl) -acetamide. The ordinate is a measure of tumor growth on day 7 compared to the vehicle control.

FIG. 3 shows inhibitor E-2-methoxy-N- (3- {4- (3-methyl-4- (6-methyl) administered PO and QD to nu / nu mice bearing SK-OV-3 tumors. The antitumor efficacy of -pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -allyl) -acetamide is shown over time. In Fig. 3, the symbols have the following meanings: circles, vehicles, BIDs; Lozenge, inhibitor at 50 mg / kg, QD; Triangle, inhibitor at 100 mg / kg, QD; And square, inhibitor at 200 mg / kg, QD.

Figure 4 shows the inhibitor E-2-methoxy-N- (3- {4- (3-methyl-4- (6-methyl) administered PO and BID to nu / nu mice bearing SK-OV-3 tumors. The antitumor efficacy of -pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -allyl) -acetamide is shown over time. In Fig. 4, the symbols have the following meanings: circles, vehicles, BIDs; Crosshair, inhibitor at 25 mg / kg, BID; Diamond, inhibitor at 50 mg / kg, BID; And asterisks, inhibitors at 100 mg / kg, BID.

5A shows inhibitor E-2-methoxy-N- (3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) administered to mice bearing BT-474 tumors. The antitumor efficacy of -phenylamino) -quinazolin-6-yl} -allyl) -acetamide is shown, illustrating the multiplicity effect of that dose.

5B shows inhibitor E-2-methoxy-N- (3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) administered to mice bearing BT-474 tumors. The antitumor efficacy of -phenylamino) -quinazolin-6-yl} -allyl) -acetamide is illustrated, illustrating the frequency (frequency) effect of that dose.

6A shows inhibitor E-2-methoxy-N- (3- {4- (3-methyl-4- (6-methyl-pyridine-3-) administered QD to mice bearing MDA-MB-453 tumors. The antitumor efficacy of yloxy) -phenylamino) -quinazolin-6-yl} -allyl) -acetamide is shown.

6B shows inhibitor E-2-methoxy-N- (3- {4- (3-methyl-4- (6-methyl-pyridine-3-) administered BID to mice bearing MDA-MB-453 tumors. The antitumor efficacy of yloxy) -phenylamino) -quinazolin-6-yl} -allyl) -acetamide is shown.

The present invention generally relates to methods of drug administration. More particularly, the present invention relates to the administration of anticancer agents, including inhibitors of the erbB2 receptor. The present invention also relates to improved methods of administering protein receptor tyrosine kinase inhibitors useful for treating abnormal cell growth, such as cancer, in mammals. The present invention also relates to kits useful for administration using said inhibitors in the treatment of abnormal cell growth in mammals, especially humans.

It is known that such cells may become cancerous by transforming a portion of the DNA of a particular cell with an oncogene, which is a gene that leads to the formation of malignant tumor cells upon activation. Many tumorigenic genes encode abnormal tyrosine kinase proteins that can cause cellular transformation. On the other hand, overexpression of normal protomatogenic genetic tyrosine kinases may lead to proliferative disorders, which often cause malignant phenotypes.

Receptor tyrosine kinases span the cell membrane; It is an enzyme that retains an extracellular binding domain for growth factors such as epidermal growth factor, transmembrane domain, and specific tyrosine residues in proteins and thus functions as a kinase that affects cell proliferation. Moreover, some receptor tyrosine kinases are substrates for the same or other protein kinases, which is a process that can modulate kinase function. Receptor tyrosine kinases fall into several classes, one of which is the erb family, including erbB1 and erbB2. It is known that kinases, such as erbB2, are often abnormally expressed in common human cancers such as breast cancer, gastrointestinal cancers such as colon, rectal or stomach cancers, leukemias, and ovarian, bronchial or pancreatic cancers. Epidermal growth factor receptor (erbB1) possessing tyrosine kinase activity, many human cancers, such as brain, lung, squamous cells, bladder, stomach, breast, head and neck, esophagus, female reproductive system and thyroid tumor It has also been found to be mutated and / or overexpressed in. Thus, it has been recognized that inhibitors of receptor tyrosine kinases are useful as selective inhibitors of cancer cell growth in mammals. Abnormal cell growth may be associated with cellular expression of the erb receptor.

However, it is not sufficiently understood that the method of administering inhibitors can affect the efficacy of such inhibitors.

The present invention generally relates to methods and kits for inhibiting abnormal cell growth. More particularly, the present invention relates to an improved dosing schedule for anticancer agents.

The present invention

(a) administering to a mammal in need thereof a therapeutically effective amount of a first inhibitor of the erbB2 receptor; And

(b) subsequent administration of said therapeutically effective amount of a second inhibitor of erbB2 receptor in one to six doses to said mammal after a certain interval comprising less than 24 hours.

Including, the present invention relates to a method for treating overexpression of an erbB2 receptor in a mammal.

In one preferred embodiment of the invention, in step (b) of the method, a therapeutically effective amount of the second inhibitor of erbB2 receptor can be administered in one to four doses. In a more preferred embodiment, in step (b) of the method, a therapeutically effective amount of the second inhibitor of erbB2 receptor can be administered in one to two doses. In another embodiment, in step (b) of the method, a therapeutically effective amount of a second inhibitor of erbB2 receptor can be administered in a single dose.

In another embodiment of the invention, the interval in step (b) of the method is less than 12 hours. In a preferred embodiment, the interval in step (b) of the method is less than 6 hours. In a more preferred embodiment, the interval in step (b) of the method is less than 3 hours. In the most preferred embodiment, the interval in step (b) of the method is less than 1 hour.

The mode of inhibitor administration in steps (a) and (b) can be oral, intraoral, sublingual, intranasal, intragastric, duodenal, topical, intraocular, rectal or vaginal.

In one aspect of the invention, the first inhibitor in step (a) is the same as the second inhibitor in step (b). In one aspect of the invention, the first dose may differ from the subsequent one to six doses. In another embodiment of the invention, the inhibitor in step (a) may be other than the inhibitor in step (b). In one specific embodiment, the inhibitor in step (a) is identical to the inhibitor in step (b), optionally in the same stereoisomer or same salt form. In another embodiment of the method of treatment, the first inhibitor in step (a) synergizes with the second inhibitor in step (b). The first inhibitor in step (a), the second inhibitor in step (b), or both can be antagonists of the erbB2 receptor.

In one aspect of the invention, the therapeutically effective amount of the first inhibitor of erbB2 receptor is different from the one to six therapeutically effective amounts of the second inhibitor of erbB2 receptor. In one preferred embodiment of the invention, the first inhibitor in step (a) is other than the second inhibitor in step (b). In another preferred embodiment, the first inhibitor in step (a) synergizes with the second inhibitor in step (b). In another preferred embodiment of the invention, the first inhibitor in step (a), the second inhibitor in step (b), or both are antagonists of the erbB2 receptor.

In one preferred embodiment of the invention, the first inhibitor in step (a) and the second inhibitor in step (b) are independently selected from small molecules and monoclonal antibodies. In one preferred embodiment, both the first inhibitor in step (a) and the second inhibitor in step (b) are small molecules or monoclonal antibodies. In another preferred embodiment of the invention, the first inhibitor in step (a), the second inhibitor in step (b), or both are selective for the erbB2 receptor.

The method of treatment of the present invention may further comprise an in vivo half-life of the inhibitor in step (a), the inhibitor in step (b), or both.

The method of the present invention may comprise administering an inhibitor in step (a), an inhibitor in step (b), or both, wherein the inhibitor is not substantially cytotoxic.

The method of the invention may comprise administering an inhibitor in step (a), an inhibitor in step (b), or both, which are substantially not mitotic inhibitors.

In one aspect of the invention, the administration is controlled release. Controlled release formulations can be administered orally, intraorally, sublingually, intranasally, gastric, duodenum, topical, intraocular, rectal or vaginal.

In one embodiment of the method of the invention, the inhibitor in step (a) and the inhibitor in step (b) are independently selected from small molecules and monoclonal antibodies. In one preferred embodiment, both the inhibitor in step (a) and the inhibitor in step (b) are small molecules or monoclonal antibodies. Such small molecules may be less than 4,000 Daltons.

The first inhibitor in step (a), the second inhibitor in step (b), or both may be selective for the erbB2 receptor.

In another embodiment of the method of treatment, the first inhibitor in step (a), the second inhibitor in step (b), or both are compounds of the formula (1), or a pharmaceutically acceptable salt, solvate or pro Contains prodrugs:

In the above formula,

m is an integer from 0 to 3;

p is an integer from 0 to 4;

R ¹ and R ² are each independently selected from H and C ₁ -C ₆ alkyl;

R ³ is-(CR ¹ R ² ) _t (4 to 10 membered heterocyclic), wherein t is an integer from 0 to 5, wherein the heterocyclic group is optionally fused with a benzene ring or a C ₅ -C ₈ cycloalkyl group Wherein the-(CR ¹ R ² ) _t -portion of the aforementioned R ³ group optionally includes a carbon-carbon double or triple bond, t is an integer from 2 to 5, and includes any of the above-mentioned fused rings; A R ³ group is optionally substituted by 1 to 5 R ⁸ groups;

R ⁴ is-(CR ¹⁶ R ¹⁷ ) _m -C≡C- (CR ¹⁶ R ¹⁷ ) _t R ⁹ ,-(CR ¹⁶ R ¹⁷ ) _m -C = C- (CR ¹⁶ R ¹⁷ ) _t -R ⁹ , -(CR ¹⁶ R ¹⁷ ) _m -C≡C- (CR ¹⁶ R ¹⁷ ) _k R ¹³ ,-(CR ¹⁶ R ¹⁷ ) _m -C = C- (CR ¹⁶ R ¹⁷ ) _k R ¹³ , or-(CR ¹⁶ R ¹⁷⁾ _t R ⁹ [wherein, the point of attachment to the R ⁹ is through a carbon atom of the R ⁹ group, k is an integer from 1 to 3, t is an integer from 0 to 5, respectively, m are each Is an integer of 0 to 3;

R ⁵ is halo, hydroxy, —NR ¹ R ² , C ₁ -C ₆ alkyl, trifluoromethyl, C ₁ -C ₆ alkoxy, trifluoromethoxy, —NR ⁶ C (O) R ¹ , -C (O) NR ⁶ R ⁷ , -SO ₂ NR ⁶ R ⁷ , -NR ⁶ C (O) NR ⁷ R ¹ , and -NR ⁶ C (O) OR ⁷ ;

R ⁶ , R ^6a and R ⁷ are each H, C ₁ -C ₆ alkyl,-(CR ¹ R ² ) _t (C ₆ -C ₁₀ aryl), and-(CR ¹ R ² ) _t (4 to 10) Circular heterocyclic), wherein t is an integer from 0 to 5, wherein one or two ring carbon atoms of the heterocyclic group are optionally substituted by an oxo (═O) moiety and R ⁶ and R described above. ^{The seven} groups of alkyl, aryl and heterocyclic moieties include halo, cyano, nitro, -NR ¹ R ² , trifluoromethyl, trifluoromethoxy, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, Optionally substituted by 1 to 3 substituents independently selected from C ₂ -C ₆ alkynyl, hydroxy and C ₁ -C ₆ alkoxy; or

R ⁶ and R ⁷ , or R ^6a and R ⁷ , when they are attached to the same nitrogen atom, may together form a 4- to 10-membered heterocyclic ring, which ring may be formed by R ⁶ , R ^6a and R ⁷ In addition to the attached nitrogen, it may comprise one to three additional hetero moieties selected from N, N (R ¹ ), O and S, provided that only two O atoms, two S atoms, or O and S atoms Should not be attached directly to each other;

R ⁸ is oxo (═O), halo, cyano, nitro, trifluoromethoxy, trifluoromethyl, azido, hydroxy, C ₁ -C ₆ alkoxy, C ₁ -C ₁₀ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, -C (O) R ⁶ , -C (O) OR ⁶ , -OC (O) R ⁶ , -NR ⁶ C (O) R ⁷ , -NR ⁶ SO ₂ NR ⁷ R ¹ , -NR ⁶ C (O) NR ¹ R ⁷ , -NR ⁶ C (O) OR ⁷ , -C (O) NR ⁶ R ⁷ , -NR ⁶ R ⁷ , -NR ⁶ OR ⁷ , -SO ₂ NR ⁶ R ⁷ , -S (O) _j (C ₁ -C ₆ alkyl), where j is an integer from 0 to 2],-(CR ¹ R ² ) _t (C ₆ -C ₁₀ aryl),-(CR ¹ R ² ) _t (4-10 membered heterocyclic),-(CR ¹ R ² ) _q C (O) (CR ¹ R ² ) _t (C ₆ -C ₁₀ aryl), -(CR ¹ R ² ) _q C (O) (CR ¹ R ² ) _t (4 to 10 membered heterocyclic),-(CR ¹ R ² ) _t O (CR ¹ R ² ) _q (C ₆ -C ₁₀ aryl),-(CR ¹ R ² ) _t O (CR ¹ R ² ) _q (4 to 10 membered heterocyclic),-(CR ¹ R ² ) _q S (O) _j (CR ¹ R ² ) _t (C ₆ -C ₁₀ aryl), and-(CR ¹ R ² ) _q S (O) _j (CR ¹ R ² ) _t (4 to 10 membered heterocyclic), wherein j is 0, 1 or 2 And q and t are each independently an integer from 0 to 5, and one or two ring carbon atoms of the heterocyclic moiety of the aforementioned R ⁸ group are optionally substituted by an oxo (═O) moiety, Alkyl, alkenyl, alkynyl, aryl and heterocyclic moieties of the group R ^{8 may be selected from the} group consisting of halo, cyano, nitro, trifluoromethyl, trifluoromethoxy, azido, -OR ⁶ , -C (O) R ⁶ , -C (O) OR ⁶ , -OC (O) R ⁶ , -NR ⁶ C (O) R ⁷ , -C (O) NR ⁶ R ⁷ , -NR ⁶ R ⁷ , -NR ⁶ OR ⁷ , C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl,-(CR ¹ R ² ) _t (C ₆ -C ₁₀ aryl), and-(CR ¹ R ² ) _t (4 To 10 membered heterocyclic) wherein t is an integer from 0 to 5 optionally substituted with 1 to 3 substituents independently selected from;

R ⁹ is a non-aromatic monocyclic ring, a fused or bridged bicyclic ring, or a spirocyclic ring, which ring contains 3 to 12 carbon atoms, of which 0 to 3 carbon atoms are N, O, S (O) _j where j is an integer from 0 to 2 and optionally substituted with a hetero moiety independently selected from -NR ^1- , provided that only two O atoms, two S (O) _j parts, O atom and S (O) _j moiety, N atom and S atom, or N atom and O atom should not be attached directly to each other in the ring, and the carbon atom of the ring is optionally substituted by 1 or 2 R ⁸ groups Become;

Each R ¹¹ is independently selected from the substituents provided within the definition of R ⁸ , provided that R ¹¹ is not oxo (═O);

R ¹² is R ⁶ , -OR ⁶ , -OC (O) R ⁶ , -OC (O) NR ⁶ R ⁷ , -OCO ₂ R ⁶ , -S (O) _j R ⁶ , -S (O) _j NR ⁶ R ⁷ , -NR ⁶ R ⁷ , -NR ⁶ C (O) R ⁷ , -NR ⁶ SO ₂ R ⁷ , -NR ⁶ C (O) NR ^6a R ⁷ , -NR ⁶ SO ₂ NR ^6a R ⁷ , -NR ⁶ CO ₂ R ⁷ , CN, -C (O) R ⁶ , or halo, j is an integer from 0 to 2;

R ¹³ is —NR ¹ R ¹⁴ or —OR ¹⁴ ;

R ¹⁴ is H, R ¹⁵ , -C (O) R ¹⁵ , -SO ₂ R ¹⁵ , -C (O) NR ¹⁵ R ⁷ , -SO ₂ NR ¹⁵ R ⁷ , or -CO ₂ R ¹⁵ ;

R ¹⁵ is R ¹⁸ ,-(CR ¹ R ² ) _t (C ₆ -C ₁₀ aryl),-(CR ¹ R ² ) _t (4-10 membered heterocyclic), wherein t is an integer from 0 to 5 Wherein one or two ring carbon atoms of the heterocyclic group are optionally substituted by an oxo (═O) moiety, wherein the aryl and heterocyclic moieties of the above R ¹⁵ groups are substituted by one to three R ⁸ substituents Optionally substituted;

R ¹⁶ and R ¹⁷ are each independently selected from H, C ₁ -C ₆ alkyl, and —CH ₂ OH, or R ¹⁶ and R ¹⁷ together are —CH ₂ CH ₂ — or —CH ₂ CH ₂ CH ₂ -take the form;

R ¹⁸ is C ₁ -C ₆ alkyl, wherein each carbon not bonded to an N or O atom, or S (O) _j , where j is an integer from 0 to 2, is optionally substituted by R ¹² ;

All of the above-mentioned substituents comprising a CH ₃ (methyl), CH ₂ (methylene), or CH (methine) group, which are not attached to a halogeno, SO or SO ₂ group, or an N, O or S atom, are hydroxy , Halo, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy and —NR ¹ R ² .

Unless otherwise indicated, the term "halo" as used herein includes fluoro, chloro, bromo or iodo. Preferred halo groups are fluoro and chloro.

Unless otherwise indicated, the term "alkyl" as used herein includes saturated monovalent hydrocarbon radicals having linear, cyclic (including mono- or multi-cyclic moieties) or branched moieties. It should be appreciated that for alkyl groups comprising cyclic moieties, they must contain at least 3 carbon atoms.

Unless otherwise indicated, the term "cycloalkyl" as used herein includes saturated monovalent hydrocarbon radicals having cyclic (mono- or multi-cyclic) moieties.

Unless otherwise indicated, the term "alkenyl" as used herein includes alkyl groups as defined above having one or more carbon-carbon double bonds.

Unless otherwise indicated, the term "alkynyl" as used herein includes alkyl groups as defined above having one or more carbon-carbon triple bonds.

Unless otherwise indicated, the term "aryl" as used herein includes organic radicals derived from aromatic hydrocarbons such as phenyl or naphthyl by removing one hydrogen.

Unless otherwise indicated, the term “alkoxy” as used herein includes —O-alkyl groups where alkyl is as defined above.

Unless otherwise indicated, the term "4-10 membered heterocyclic" as used herein includes aromatic and non-aromatic heterocyclic groups containing one or more heteroatoms each selected from O, S and N. And each heterocyclic group has 4 to 10 atoms in its ring system. Non-aromatic heterocyclic groups include groups having only 4 atoms in their ring system, but aromatic heterocyclic groups must have at least 5 atoms in their ring system. Such heterocyclic groups include benzo fused ring systems, and ring systems substituted by one or more oxo moieties. One example of a four-membered heterocyclic group is azetidinyl (derived from azetidine). One example of a 5 membered heterocyclic group is thiazolyl and one example of a 10 membered heterocyclic group is quinolinyl. Examples of non-aromatic heterocyclic groups include pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxa Neil, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazinyl, diazepinyl, thiazinyl, 1,2,3,6-tetra Hydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, ditianyl, dithiola Nyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo [3.1.0] hexanyl, 3-azabicyclo [4.1. 0] heptanyl, 3H-indolyl and quinolizinyl. Examples of aromatic heterocyclic groups include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, Pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolinyl, phthalazinyl, pyridazinyl, triazinyl, isoindoleyl, Putridinyl, furinyl, oxdiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl and furopyridinyl . The aforementioned groups as derived from the compounds listed above may be C-attached or N-attached if possible. For example, the group derived from pyrrole may be pyrrole-1-yl (N-attached) or pyrrole-3-yl (C-attached).

The term "Me" means methyl, "Et" means ethyl and "Ac" means acetyl.

Unless otherwise indicated, "pharmaceutically acceptable salt (s)" as used herein includes salts of acidic or basic groups that may be present in the compounds of the present invention. Compounds of the invention which are basic in nature are capable of forming a wide range of salts with various inorganic and organic acids. Acids that can be used to prepare pharmaceutically acceptable acid addition salts of these basic compounds are non-toxic acid addition salts, ie salts containing pharmacologically acceptable anions, such as hydrochloride, hydrobromide, hydro Iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, as Corbate, succinate, maleate, gentinate, fumarate, gluconate, glucuronate, sacrate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- Toluenesulfonate and pamoate [ie, 1,1'-methylene-bis- (2-hydroxy-3-naphthoate)] salt Will. Compounds of the invention comprising a basic moiety such as an amino group may form pharmaceutically acceptable salts with various amino acids other than the acids mentioned above.

The method of treatment of the invention comprises an inhibitor in step (a), an inhibitor in step (b), or both, gefitinib (IRESSA, ZD1839), trastuzumab, cetuximab , Erlotinib, IDM-1, ABX-EGF, canertinib hydrochloride, EGF-P64k vaccine, EKB-569, EMD-72000, GW-572016, MDX-210, ME-103 , YMB-1001, 2C4 antibody, APC-8024, CP-724714, E75, Her-2 / neu vaccine, Herzyme, TAK-165, ADL-681, B-17, D-69491, Dab-720 , EGFrvIII, EHT-102, FD-137, HER-1 vaccine, HuMax-DGFr, ME-104, MR1-1, SC-100, trastuzumab-DM1, YMB-1005, AEE-788 (Novartis), mTOR Administering an erbB2 receptor comprising a compound selected from the group consisting of inhibitors (including Rapamycin (Rapamune, Siolimus, Wyeth)), CCI-779 (Wyeth), AP23573 (ARIAD) and RAD001 (Novartis) Can be.

In one embodiment of the present invention, an erbB2 receptor is employed, using cytogenetic testing, fluorescence in situ hybridization measurement, immunohistochemistry testing, flow cytometric testing, tests based on reverse transcriptase polymerase chain reaction, or any combination thereof. Determine overexpression of.

In one aspect of the invention, the mammal is a human and the abnormal cell growth is cancer. Mammals may be laboratory animals, domestic pets, farm animals, or any other animal.

The method of treatment of the present invention may further comprise reaching plasma levels of the first inhibitor in step (a), the second inhibitor in step (b), or both, from 10 ng / ml to 4000 ng / ml. Can be.

In one embodiment of the invention, the first inhibitor in step (a) and the second inhibitor in step (b) are each independently selected from the group consisting of:

(±)-(3-methyl-4- (pyridin-3-yloxy) -phenyl)-(6-piperidin-3-ylethynyl-quinazolin-4-yl) -amine;

(+)-(3-methyl-4- (pyridin-3-yloxy) -phenyl)-(6-piperidin-3-ylethynyl-quinazolin-4-yl) -amine;

(-)-(3-methyl-4- (pyridin-3-yloxy) -phenyl)-(6-piperidin-3-ylethynyl-quinazolin-4-yl) -amine;

2-methoxy-N- (3- {4- (3-methyl-4- (pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -prop-2-ynyl) -acetyl amides;

(±)-(3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenyl)-(6-piperidin-3-ylethynyl-quinazolin-4-yl) -amine;

(+)-(3-Methyl-4- (6-methyl-pyridin-3-yloxy) -phenyl)-(6-piperidin-3-ylethynyl-quinazolin-4-yl) -amine ;

(-)-(3-Methyl-4- (6-methyl-pyridin-3-yloxy) -phenyl)-(6-piperidin-3-ylethynyl-quinazolin-4-yl) -amine;

2-methoxy-N- (3- {4- (3-methyl-4- (2-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -prop-2- Phosphoryl) -acetamide;

(3-Methyl-4- (2-methyl-pyridin-3-yloxy) -phenyl)-(6-piperidin-4-ylethynyl-quinazolin-4-yl) -amine;

(3-Methyl-4- (6-methyl-pyridin-3-yloxy) -phenyl)-(6-piperidin-4-ylethynyl-quinazolin-4-yl) -amine;

2-methoxy-N- (3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -prop-2- Phosphoryl) -acetamide;

2-Fluoro-N- (3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -prop-2- Phosphoryl) -acetamide;

E-2-methoxy-N- (3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -allyl)- Acetamide;

(3-Methyl-4- (pyridin-3-yloxy) -phenyl)-(6-piperidin-4-ylethynyl-quinazolin-4-yl) -amine;

2-methoxy-N- (1- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-ylethynyl} -cyclopropyl) Acetamide;

EN- (3- {4- (3-Chloro-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -allyl) -2-methoxy-acetamide ;

N- (3- {4- (3-Chloro-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -prop-2-ynyl) -acetamide ;

N- (3- {4- (3-Methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -prop-2-ynyl) -acetamide ;

E-N- (3- {4- (3-Chloro-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -allyl) -acetamide;

E-2-ethoxy-N- (3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -allyl)- Acetamide;

1-Ethyl-3- (3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -prop-2-ynyl ) -Urea;

Piperazine-1-carboxylic acid (3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -prop-2-ynyl )-amides;

(±) -2-hydroxymethyl-pyrrolidine-1-carboxylic acid (3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin- 6-yl} -prop-2-ynyl) -amide;

(+)-2-hydroxymethyl-pyrrolidine-1-carboxylic acid (3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin- 6-yl} -prop-2-ynyl) -amide;

(-)-2-hydroxymethyl-pyrrolidine-1-carboxylic acid (3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin- 6-yl} -prop-2-ynyl) -amide;

2-Dimethylamino-N- (3- {4- (3-methyl-4- (pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -prop-2-ynyl) -acetyl amides;

E-N- (3- {4- (3-Methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -allyl) -methanesulfonamide;

Isoxazole-5-carboxylic acid (3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -prop-2-ynyl )-amides;

1- (1,1-dimethyl-3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -prop-2 -Personal) -3-ethyl-urea.

The method of treatment of the present invention includes the use of a single agent that inhibits the erbB2 receptor as well as the use of two different agents. Such a single agent and at least one of the two agents is preferably an agent according to formula (1). Thus, in one embodiment, the inhibitor is (±)-(3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenyl)-(6-piperidin-3-ylethynyl-quinazoline -4-yl) -amine; And pharmaceutically acceptable salts, prodrugs and solvates thereof. In another embodiment, the inhibitor is (3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenyl)-(6-piperidin-4-ylethynyl-quinazolin-4-yl) Amines; And pharmaceutically acceptable salts, prodrugs and solvates thereof. In another embodiment, the inhibitor is E-2-methoxy-N- (3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6 -Yl} -allyl) -acetamide; And pharmaceutically acceptable salts, prodrugs and solvates thereof. In still another embodiment, the inhibitor is EN- (3- {4- (3-chloro-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -allyl ) -2-methoxy-acetamide; And pharmaceutically acceptable salts, prodrugs and solvates thereof. In another embodiment, the inhibitor is EN- (3- {4- (3-chloro-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -allyl)- Acetamide; And pharmaceutically acceptable salts, prodrugs and solvates thereof. In certain embodiments of the invention, the inhibitor is piperazine-1-carboxylic acid (3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6- Yl-prop-2-ynyl) -amide; And pharmaceutically acceptable salts, prodrugs and solvates thereof. In another specific embodiment of the invention, the inhibitor is EN- (3- {4- (3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino) -quinazolin-6-yl} -Allyl) -methanesulfonamide; And pharmaceutically acceptable salts, prodrugs and solvates thereof. In another aspect of the invention, the first inhibitor of step (a), the second inhibitor of step (b), or both are in a pharmaceutically acceptable carrier.

In one embodiment of the present invention, abnormal cell growth occurs due to overexpression of the erbB2 receptor. Abnormal cell growth treated with the first and second erbB2 receptor inhibitors may be cancer. These cancers include terminal acute melanoma, actinic keratosis, adenocarcinoma, adenocystic carcinoma, adenocarcinoma, adenocarcinoma, glandular squamous cell carcinoma, astrocytoma, bartholin gland carcinoma, basal cell carcinoma, bronchial adenocarcinoma, capillaries Carcinoma, carcinoid, carcinoma, carcinosarcoma, cavernous carcinoma, cholangiocarcinoma, chondrosarcoma, choroid plexus papilloma, choroid plexus carcinoma, clear cell carcinoma, cyst adenocarcinoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrium Sheep adenocarcinoma, ventricular carcinoma, epithelial carcinoma, Ewing's sarcoma, fibrolaminar carcinoma, focal nodular hyperplasia, gastrinoma, germ cell tumor, glioblastoma, glucagon, hemangioblastoma, hemangioma, hemangioma , Hepatic adenoma, hepatic adenopathy, hepatocellular carcinoma, insulin carcinoma, intraepithelial tumor, hepatic squamous cell tumor, invasive squamous cell carcinoma, giant cell carcinoma, leiomyosarcoma, malignant melanoma, malignant Melanoma, malignant mesothelioma, medulloblastoma, medulla, melanoma, meninges, mesothelioma, metastatic carcinoma, myxoid epithelial carcinoma, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, oat cell carcinoma, oligodendrocyte, osteosarcoma , Pancreatic polypeptide, papillary serous adenocarcinoma, pineal gland cell, pituitary tumor, plasmacytoma, pseudosarcoma, lung blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin -Secretory tumors, squamous carcinoma, squamous cell carcinoma, subcutaneous, shallow diffuse malignant melanoma, undifferentiated carcinoma, uveal melanoma, warty carcinoma, VIP species, well differentiated carcinoma, bronchoalveolar cell carcinoma (BAC) and Wilm tumor (Wilm's tumor) can be selected from the group consisting of.

In one embodiment, the abnormal cell growth is a tumor selected from the group consisting of lung, breast, skin, stomach, intestine, esophagus, pancreas, liver, bladder, head, neck, brain, cervix, and ovarian tumors. In one preferred embodiment, the abnormal cell growth is a tumor selected from the group consisting of breast, stomach, pancreatic and ovarian tumors. In a more preferred embodiment, the abnormal cell growth is breast cancer.

In another embodiment of the invention, the erbB2 receptor inhibitor may be selective for the erbB2 receptor. The method of the present invention comprises the steps of (c) calculating the ratio of the binding affinity of the inhibitor to the erbB2 receptor and the second binding affinity of the inhibitor to the erbB1 receptor; And (d) assessing selectivity using this ratio. In one embodiment, the inhibitor is at least 2-fold selective for the erbB2 receptor. In another embodiment, the inhibitor is at least 10-fold selective for the erbB2 receptor.

In another embodiment, the invention provides a first dose of an erbB2 receptor inhibitor, a therapeutically synergistic second effective amount of such inhibitor, and a third or fourth dose of any of the above inhibitors, within a 24 hour period, in response to abnormal cell growth. A method of treating a subject exhibiting abnormal cell growth, including oral, intraoral, sublingual, intranasal, intraocular, gastric, duodenal, topical, rectal or vaginal administration to a subject in need thereof. The inhibitor may be a selective erbB2 receptor inhibitor.

In another aspect of the invention, the invention provides a dose of at least two doses of an erbB2 receptor inhibitor that is suitable for oral, buccal, sublingual, intranasal, intraocular, gastric, duodenal, topical, rectal or vaginal administration to a particular subject. And a kit for treating abnormal cell growth, including written instructions recommending that such dose be administered to a subject exhibiting abnormal cell growth at least twice daily. Advantageously, these written instructions are on the label or package insert. In one embodiment of the kit of the invention, the abnormal cell growth is a tumor selected from the group consisting of lung, breast, skin, stomach, intestine, esophagus, bladder, head, neck, brain, cervix, and ovarian tumor.

In another embodiment of the invention, the invention is administered by injecting a therapeutically effective amount of an erbB2 receptor inhibitor over 1 to 8 hours into a subject in need of tumor treatment (since such infusion is more effective than bolus injection). Including, a method of treating a tumor comprising an erbB2 receptor in said subject. Such infusions can be intravenous, intramuscular, intraperitoneal or subcutaneous routes. In one embodiment, the inhibitor may be a compound according to formula (1).

In another aspect of the invention, the invention comprises the steps of (a) determining a reference dose of an erbB2 receptor inhibitor; And (b) dividing this dose to increase potency, thereby enhancing the potency of the erbB2 receptor inhibitor in the particular subject. Increased potency is a form of synergy resulting from dividing the dose. In one embodiment, this dose is divided into two to six doses per day.

In another embodiment, the reference dose exhibits side effects, and divided doses reduce these side effects. Inhibitors may be at least about two times selective for the erbB2 receptor as compared to the erbB1 receptor. In another embodiment, the inhibitor may be at least 10-fold selective for the erbB2 receptor as compared to the erbB1 receptor.

The method of enhancing efficacy may comprise (c) estimating the ratio of the binding affinity of the inhibitor to the erbB2 receptor and the second binding affinity of the inhibitor to the erbB1 receptor; And (d) assessing selectivity using this ratio.

In another aspect of the invention, the invention comprises administering a therapeutically effective amount of an inhibitor to a patient in need thereof, wherein the plasma level of the inhibitor in said patient is a single daily To be lower than the dose, the daily dose is divided, thereby increasing efficacy), and methods of increasing the efficacy of the erbB2 receptor inhibitor.

In another embodiment, the invention provides a method for enhancing safety following administration of an erbB2 receptor inhibitor in such subject, comprising administering to the subject in need thereof a therapeutically effective amount of the erbB2 receptor inhibitor in two to six doses per day. It includes.

In another embodiment, the present invention provides safety following administration of an erbB2 receptor inhibitor in a particular subject, including determining a daily baseline dose of the erbB2 receptor inhibitor having a safety profile and dividing the dose to enhance this safety profile. It includes a method of enhancing.

In another embodiment, the present invention provides a dose of an erbB2 receptor inhibitor, a dose suitable for intravenous, intramuscular, intraperitoneal, or subcutaneous infusion, and written instructions recommending that the subject be infused over 1 to 8 hours. And a kit for treating abnormal cell growth in said subject. In one embodiment of this kit, abnormal cell growth is associated with a tumor selected from the group consisting of lung, breast, skin, stomach, intestine, esophagus, bladder, pancreas, liver, head, cervix, brain, cervix, and ovarian tumors. There may be.

In another aspect, the invention includes a method of prophylactic treatment for a subject at risk of tumor development, including administering an effective amount of a selective inhibitor of the erbB2 receptor to a subject at risk of tumor development at least twice a day. do. In one embodiment of such prophylactic methods of treatment, the inhibitor may be other than an antibody or fragment thereof.

In another embodiment, the invention comprises administering a therapeutically effective amount of a erbB2 receptor inhibitor to a patient in need thereof, wherein the plasma level of the inhibitor in the patient is a therapeutically effective amount of a single daily dose To be lower, the daily dose is divided, thereby increasing potency), and a method of increasing the potency of the erbB2 receptor inhibitor. In one embodiment, plasma levels are expressed as Cave. In another embodiment, plasma levels are expressed as C _max . The inhibitor may be a selective erbB2 receptor inhibitor. In one embodiment, the inhibitor may be other than an antibody or fragment thereof.

In another embodiment, the invention comprises administering a therapeutically effective amount of an erbB2 receptor inhibitor to a subject in need of tumor treatment over 1 to 8 hours (since such infusion is more effective than bolus injection), And a method of treating a tumor comprising an erbB2 receptor in said subject. By cyclic injection means relatively rapid therapeutic injection depending on the nature of the injection site. The infusion can be an intravenous, intramuscular, intraperitoneal or subcutaneous route. The subject of the method of treatment may be a human, but any mammal is suitable. In one embodiment, the tumor is cancer. In the method of the invention, the injection can be characterized as being performed at a non-uniform rate. For example, the rate of administration may be increased or slowed during the infusion. The inhibitor may be selective for the erbB2 receptor. Moreover, the method includes estimating the ratio of the binding affinity of the inhibitor to the erbB2 receptor and the second binding affinity of the inhibitor to the erbB1 receptor; And assessing selectivity using this ratio. Other methods known in the art are also suitable for evaluating selectivity. In one embodiment, the inhibitor is at least 2-fold selective for the erbB2 receptor. In another embodiment, the inhibitor is at least 10-fold selective for the erbB2 receptor. The subject of the method of treatment of the invention may be a human. The inhibitor may be an antagonist. In one embodiment, the inhibitor is other than an antibody or fragment thereof. In particular, the inhibitor may be a small molecule. The method of the present invention may further comprise an in vivo half life of the inhibitor from 30 minutes to 8 hours.

In one aspect, the present invention

(a) determining overexpression of the erbB2 receptor using cytogenetic testing, fluorescence in situ hybridization, immunohistochemical testing, flow cytometric testing, reverse transcriptase polymerase chain reaction, or a combination test thereof;

(b) administering to the mammal a therapeutically effective amount of a first inhibitor of erbB2 receptor based on overexpression of the erbB2 receptor from step (a); And

(c) subsequent administration of said therapeutically effective amount of said second inhibitor of erbB2 receptor in 1 to 6 doses to said mammal based on overexpression of erbB2 receptor from step (a) after a certain interval comprising less than 24 hours.

Including, the present invention relates to a method for treating overexpression of an erbB2 receptor in a mammal.

The method may comprise the injection of an inhibitor that is not substantially cytotoxic. The method may also include the injection of an inhibitor that is substantially not a mitosis inhibitor.

The method of treatment by infusion of the inhibitor may further comprise that such infusion is at least 20% more potent than the bolus injection.

The method of treatment by infusion may further comprise infusion twice or three times daily.

The method of treatment by infusion may further comprise reaching the plasma level of the inhibitor from 10 ng / ml to 4000 ng / ml.

Unless otherwise indicated, the term “treating” as used herein refers to reversing, alleviating, inhibiting or preventing the progression of the disorder or disease to which such term applies, or one or more symptoms of such disorder or disease. Means that. Unless otherwise indicated, the term "treatment" as used herein refers to the "treating" action as defined immediately above.

Unless otherwise indicated, the term “C _max ” as used herein refers to the maximum concentration in a blood, serum or plasma of such agent after administration of that particular agent. The agent is typically an erbB2 receptor inhibitor according to Formula 1.

Unless otherwise indicated, the term "AUC" as used herein refers to the area under the curve, which is a measure of the concentration of the integrated agent over time.

Unless otherwise indicated, the terms "Cave" or "C _ave " as used herein mean a measure of the average concentration of an agent over a defined time.

Unless otherwise indicated, the term "PK" as used herein refers to the distribution or pharmacokinetics of an agent over time.

Unless otherwise indicated, the terms “QD” and “BID” as used herein refer to daily and twice daily administration, respectively.

Unless otherwise indicated, the term "p.o." as used herein. And "i.v." mean oral and intravenous routes of administration, respectively.

Unless otherwise indicated, the term "PD" as used herein refers to pharmacodynamics which is the result of a functional analysis of a particular agent.

Unless otherwise indicated, the term “selectivity” as used herein refers to efficacy compared to another agent, which is usually presented as a ratio of the inhibition constant (eg IC value as IC ₅₀ ). do. On the other hand, the selectivity can be measured as the affinity of the inhibitor for the erbB2 receptor compared to the affinity for another receptor, for example erbB1. Selectivity includes any conventional manner known in the art, including, but not limited to, absolute potency, potency compared to another agent, potency compared to another agent, and presence or degree of non-erbB2 receptor effect. Can be measured.

Unless otherwise indicated, the term “inhibiting the erbB2 receptor” as used herein refers to competitively or non-competitively blocking binding of an activator that is an agonist, translocating a bound activator, or Reducing affinity constants, increasing the release rate of the activator, dissociating multimeric receptors, agglomerating monomeric receptors, or lowering the intracellular metabolic influence of receptor activation.

Unless otherwise indicated, the term "synergism" or "synergistic" as used herein means that the combined effect of the two inhibitors is greater than the sum of the effects of each inhibitor alone.

Unless otherwise indicated, the term "agonist" as used herein refers to a drug that binds to physiological receptors and mimics the effects of endogenous modulating compounds. Unless otherwise indicated, the term "antagonist" as used herein means a drug that binds to a specific receptor and does not mimic the binding of an endogenous agonist. A drug or compound that lacks its own regulatory activity but exerts effect by inhibiting the action of an agonist is termed an "antagonist".

Unless otherwise indicated, the term "side effect" as used herein means a drug action or effect other than the desired effect.

Unless otherwise indicated, the term "reduction of side effects" as used herein means reduced drug action or effects other than the desired effect.

Unless otherwise indicated, the term "inhibitor" as used herein refers to a chemical substance that stops the activity of an enzyme or receptor.

Compounds of formula (I), which are inherently acidic, may form base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts of the compounds of the invention, in particular calcium, magnesium, sodium and potassium salts.

Certain functional groups contained in the compounds of the present invention are groups that have similar spatial or electronic requirements as bioisosteric groups, i.e., parent groups, but differ from or in terms of physicochemical or other properties. Can be substituted. Suitable examples are well known to those of skill in the art and include those described in Patini et al., Chem. Rev, 1996, 96, 3147-3176, and references cited therein, including but not limited to.

Compounds of formula (1) may have asymmetric centers and therefore exist in different enantiomeric and diastereomeric forms. The present invention relates to the use of all optical isomers and stereoisomers of the compounds of the present invention, and mixtures thereof; And all pharmaceutical compositions and methods of treatment that can utilize or contain them. The compound of formula 1 may also exist as a tautomer. The present invention relates to the use of all such tautomers and mixtures thereof.

The invention also relates to the same isotopes as recited in Formula 1, except that one or more atoms are replaced by an atom having an atomic mass or mass number that is different from the atomic mass or mass number normally found in nature. Labeled compounds, and the use of pharmaceutically acceptable salts, solvates and prodrugs thereof. Examples of isotopes that can be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, for example ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, respectively. , ¹⁸ 0, ¹⁷ O, ³⁵ S, ¹⁸ F, and ³⁶ Cl. Compounds of the present invention, prodrugs thereof, and pharmaceutically acceptable salts of such compounds or prodrugs containing the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the present invention. Certain isotopically-labelled compounds of the invention, such as those incorporating radioisotopes such as ³ H and ¹⁴ C, are useful for drug and / or substrate tissue distribution assays. Tritium treated, ie ³ H and carbon-14, ie ¹⁴ C isotopes are particularly preferred in terms of ease of preparation and detectability. In addition, substitutions with heavier isotopes, such as deuterium, i.e. ² H, may be of particular therapeutic benefit resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage required, etc. May be desirable under some circumstances. Isotope-labeled compounds of formula (1) and prodrugs thereof of the present invention are generally substituted by isotopically-labeled reagents with readily available isotopically-labeled reagents, thereby providing the following schemes and / or examples and It can be prepared by carrying out the procedure described in the preparation examples.

Compounds of formula (I) having free amino, amido, hydroxy or carboxyl groups can be converted to prodrugs. Prodrugs include covalently linked amino acid residues or polypeptide chains of two or more (eg 2, 3 or 4) amino acid residues to the free amino, hydroxy or carboxylic acid groups of the compound of formula 1 via amide or ester linkages. Compounds linked with are included. Amino acid residues include, but are not limited to, 20 natural amino acids commonly designated as 3-letter symbols, and also include 4-hydroxyproline, hydroxylysine, democin, isodemosin, 3-methylhistidine, norvaline , Beta-alanine, gamma-aminobutyric acid, citrulline homocysteine, homoserine, ornithine and methionine sulfone. Additional types of prodrugs are also encompassed. For example, free carboxyl groups can be derivatized as amides or alkyl esters. Free hydroxy groups include, but are not limited to, hemisuccinate, phosphate esters, dimethylaminoacetate, and phosphoryloxymethyloxycarbonyl, as summarized in Advanced Drug Delivery Reviews, 1996, 19, 115. Derivatization may be carried out using non-limiting groups. Carbamate prodrugs of hydroxy and amino groups are also included, as are carbonate prodrugs, sulfonate esters and sulfate esters of hydroxy groups. The hydroxy group is (acyloxy) methyl and (acyloxy) ethyl ether, wherein the acyl group may be an alkyl ester optionally substituted by a group including but not limited to ether, amine and carboxylic acid functional groups, or the acyl group may be Derivatizing the same amino acid ester] is also encompassed. Prodrugs of this type are described in J. Med. Chem. 1996, 39, 10. Free amines may also be derivatized as amides, sulfonamides or phosphonamides. In both of these prodrug moieties, groups may be incorporated, including but not limited to ether, amine and carboxylic acid functional groups.

The method of the present invention may comprise administering an inhibitor in step (a), an inhibitor in step (b), or both, wherein the inhibitor is not substantially cytotoxic. Cytotoxicity can be determined by any means conventional in the art, including but not limited to measurement of apoptosis and metabolic functions such as respiratory action and substrate utilization. Substantially, cytotoxicity will be understood by those skilled in the art that, in the present invention, cytotoxicity is generally found upon use in an in vitro assay or upon administration of the agent to a test animal under conditions and concentrations corresponding to the use of the agent. it means.

The method of the invention may comprise administering an inhibitor in step (a), an inhibitor in step (b), or both, which are not substantially mitotic inhibitors. Mitosis can be determined by any means conventional in the art, including but not limited to the determination of mitosis index, DNA content and cell number. Substantially mitotic inhibitors will be understood by those skilled in the art that, in the present invention, mitotic reduction is generally found upon use in an in vitro assay or upon administration of the agent to a test animal under conditions and concentrations corresponding to the use of the agent. Means that.

In vitro activity of a compound for use in the methods of the invention can be determined by the amount of phosphorylation inhibition by the test compound compared to the control. Recombinant erbB2 (amino acid residues 675-1255) and EGFR (amino acid residues 668-1211) intracellular domains were expressed as GST fusion proteins in Baculovirus -infected Sf9 cells, which were subjected to affinity chromatography on glutathione sepharose beads Purification was carried out. Phosphorylation of poly (Glu, Tyr) is described in JD Moyer, EG Barbacci, KK Iwata, L. Arnold, B. Boman, A. Cunningham, et al., Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase, Cancer Res . 57 (1997) 4838-4848, except that the kinase reaction was 50 μl of 50 mM HEPES, pH 7.4 containing 125 mM sodium chloride, 10 mM magnesium chloride, 0.1 mM sodium orthovanadate and 1 mM ATP. Was performed in.

Tyrosine phosphorylation in native cells can be measured using the following assay. Transfected with human EGFR or BD Cohen, DR Lowy, JT Schiller, Transformation-specific interaction of the bovine papillomavirus E5 oncoprotein with the platelet-derived growth factor receptor transmembrane domain and the epidermal growth factor receptor cytoplasmic domain, J. Virol. , 67 (1993) 5303-5311], or NIH3T3 cells transfected with chimeric receptors with EGFR extracellular domain and erbB2 intracellular domain were seeded into 96 well tissue culture plates in DMEM [F. Fazloli, UH]. Kim, SG Rhee, CJ Molloy, O. Segatto, PP DiFiore, The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency, Mol . Cell . Biol . , 11 (1991) 2040-2048.

An inhibitor in DMSO (or DMSO vehicle in the control group) was added 24 hours after plating and incubated with the cells for 2 hours at 37 ° C. Cells were stimulated with human recombinant EGF (50 ng / ml final concentration) for 15 minutes at room temperature. The medium was aspirated and cells were fixed for 30 minutes with 100 μl of cold 1: 1 ethanol: acetone containing 200 μM Na ₃ VO ₄ . Plates were washed with wash buffer (0.5% Tween-20 in PBS) and 100 μl blocking buffer (3% bovine serum albumin in PBS + 200 μM fresh sodium orthovanadate) was added. The plates were further incubated for 1 hour at room temperature and washed twice with wash buffer. Anti-phosphotyrosine antibody (PY54) labeled with horseradish peroxidase was added to the wells and incubated for 1 hour at room temperature. The antibody was removed by aspiration and the plates washed four times with wash buffer. The colorimetric signal was developed by adding TMB microwell peroxidase substrate (Kirkegaard and Perry, Gaithersburg, MD) at 50 μl per well and stopped by adding 0.09 M sulfuric acid at 50 μl per well. Phosphotyrosine is evaluated by measuring absorbance at 450 nm. Signals from control wells containing no compounds stimulated with EGF after subtracting background from wells containing no EGF were defined as 100% of control. Examination of extracts from these EGF-stimulated cells by Western blotting with anti-phosphotyrosine showed that the majority of protein phosphotyrosine showed autophosphorylated EGFR or EGFR / erbB2 chimeras, respectively, but other proteins. Substrates also indicated increased tyrosine phosphorylation. EGF typically increased approximately 4-fold total phosphotyrosine levels in each transfected cell. IC ₅₀ values represent the concentration of compound required to reduce the signal to 50% of the control, which was determined graphically by titration over a 100-fold concentration range. ErbB phosphorylation was analyzed by immunoblotting and western blotting. SKBr3 cells were treated with the compound or activating ligand as indicated. The medium was aspirated and buffered for 1 ml / 75 cm 2 flask ice cold immunoprecipitation lysis [1.0% TX100 per 50 ml buffer; 10 mM Tris; 5 mM EDTA; 50 mM NaCl; Newly added 30 mM sodium orthovanadate, and 1 Complete ™ protease inhibitor tablet (Roche Diagnostics, Indianapolis, IN) with 100 μM PMSF was added. Immunoprecipitation was performed on 100 μl of lysate: EGFr was immunoprecipitated using Santa Cruz SC-120, 2 μg / 100 μl lysate; Immunoprecipitate erbB2 using the tumorigenic gene OP15, 1 μg / 100 μl lysate; ErbB3 was immunoprecipitated using Santa Cruz SC-285, 2 μg / 100 μl lysate. All immunoprecipitations were performed overnight at 4 ° C. with vibration in the presence of 30 μl of Protein A beads. Beads with immobilized protein were separated by centrifugation at 14,000 rpm for 10 seconds at 4 ° C. The supernatant was aspirated and the pellet washed three times with PBS with 0.1% Tween 20. The samples were then resuspended in 40 μl Laemmli buffer with DTT and boiled for 4 minutes. The sample was then loaded on 4-12% PAGE. They were electrophoresed for one hour at 150V using MES buffer. The gel was transferred to PVDF in the presence of 10% methanol. Block the membrane using blocking buffer (Roche Diagnostics, Indianapolis, IN), detect phosphotyrosine using an anti-PY54 antibody conjugated to horseradish peroxidase, and follow the manufacturer's instructions (ECL ™; Development was by enhanced chemiluminescence according to Amersham, Pharmacia Biotech, Piscataway, NJ; LumiGLO ™; Cell Signaling). Signals were quantified using Lumi-Imager ™ (Boehringer Mannheim, Indianapolis, IN).

The following assays can also be used for c-erbB2 kinases to determine the potency and selectivity of the compounds for their use as c-erbB2 inhibitors. The following assays are described in Schrang et. al. Anal. Biochem. 211, 1993, p233-239. Nunc MaxiSorp 96-well plates with 100 ml of 0.25 mg / mL poly (Glu, Tyr) 4: 1 (PGT) per well in PBS (phosphate buffered saline) (Sigma Chemical Co., St. Louis, MO) Coated by incubating overnight at 37 ° C. Excess PGT was aspirated off and the plate washed three times with wash buffer (0.1% Tween 20 in PBS). In 50 mL of 50 mM HEPES (pH 7.5) containing 125 mM sodium chloride, 10 mM magnesium chloride, 0.1 mM sodium orthovanadate, 1 mM ATP, 0.48 mg / mL (24 ng / well) c-erbB2 intracellular domain Perform the kinase reaction. The intracellular domain of erbB2 tyrosine kinase (amino acids 674-1255) was expressed as a GST fusion protein in baculovirus and purified by binding to and eluting glutathione coated beads. Compounds in DMSO (dimethylsulfoxide) were added to give a final DMSO concentration of 2.5%. Phosphorylation was initiated by the addition of ATP (adenosine triphosphate), which was run for 6 minutes at room temperature with constant shaking. The kinase reaction is terminated by aspirating the reaction mixture and subsequent washing with wash buffer (see above). 50 mL of HRP-conjugated PY54 (Oncogene Science Inc. Uniondale, NY) anti-phosphotyrosine antibody diluted at 0.2 mg / mL in blocking buffer (0.05% Tween 20 and 3% BSA in PBS) Phosphorylated PGT is measured by incubation for 25 minutes with. The antibody is aspirated off and the plate washed four times with wash buffer. The colorimetric signal was developed by adding TMB microwell peroxidase substrate (Kirkegaard and Perry, Gaithersburg, MD) at 50 mL per well and stopped by adding 0.09 M sulfuric acid at 50 mL per well. Phosphotyrosine is evaluated by measuring absorbance at 450 nm. The signal for the control is typically between 0.6 and 1.2 absorbance units, which is proportional to the incubation time for 10 minutes with little background in wells without PGT substrate. Inhibitors were identified by the fact that the signal was reduced compared to wells without inhibitors, and IC ₅₀ values corresponding to the concentration of compound required to inhibit 50% were determined. Compounds exemplified herein, corresponding to Formula 1, have an IC ₅₀ value of <10 mM against erbB2 kinase. IC ₅₀ values can be used to determine selectivity by any means known in the art. For example, a ratio of IC ₅₀ values at the erbB1 receptor and the erbB2 receptor (IC ₅₀ erbB1 ÷ IC ₅₀ erbB2) can be used. Advantageously the ratio is greater than two.

In vivo antitumor activity of a compound for use in the methods of the invention can be determined by the amount of inhibition of tumor growth by the test compound as compared to the control. The tumor growth inhibitory effects of various compounds are described in Corbett TH, et al., "Tumor Induction Relationships in Development of Transplantable Cancers of the Colon in Mice for Chemotherapy Assays, with a Note on Carcinogen Structure", Cancer Res., 35 , 2434-2439 (1975) and Corbett TH, et al., "A Mouse Colon-tumor Model for Experimental Therapy", Cancer Chemother. Rep. (Part 2) ", 5, 169-186 (1975)] can be measured using a slight modification. Tumors are subcutaneous from 1 to 5 million log cultured tumor cells suspended in 0.1 ml RPMI 1640 medium. (sc) can be induced in the left flank of the mouse, after sufficient time has elapsed (about 100 to 150 mm 3/5 to 6 mm in diameter) enough to promote (touch by hand) the tumor, Test animals (thymic female mice) were formulated with test compounds (formulated at concentrations of 10-15 mg / ml in 5 Gelucire or 0.5% methyl cellulose) once or twice daily for 7 to 29 days consecutively (iv Or by oral (po) administration To determine antitumor effects, tumors were measured in millimeters using Vernier calipers across two diameters, and described in Geran, RI, et. al. "Protocols for Screening Chemical Agents and Natural Products Agains Tumor size (VIII) is calculated using the following equation according to the method of t Animal Tumors and Other Biological Systems ", Third Edition, Cancer Chemother. Rep., 3, 1-104 (1972). = (W x W) / 2 x L (L = Length and W = Width) The result is an equation: Growth Inhibition Rate (%) = [100-{(% growth in treatment group /% growth in control) x 100}] The flank area where the tumor is implanted provides a reproducible dose / response effect for various chemotherapeutic agents, and the measurement method (tumor diameter) is a reliable method for assessing tumor growth rate.

Administering the erbB2 inhibitor can be carried out by any method that enables delivery of the compound to the site of action. These methods include oral route, duodenal route, parenteral injection (including intravenous, subcutaneous, intramuscular, endovascular or infusion), topical and rectal administration.

The amount of active compound administered will depend on the subject to be treated, the severity of the disorder or disease, the rate of administration, the nature of the compound, and the discretion of the prescribing physician. However, the effective dosage is in the range of 0.001 to 200 mg / kg body weight per day, preferably 1 to 35 mg / kg per day. In the case of a 70 kg human, this effective dose will be 0.05-7 g per day, preferably 0.2-2.5 g per day. In some cases, dosage levels below the lower limit of the aforementioned ranges may be more suitable, but in other cases larger doses may be used that do not cause deleterious side effects.

The erbB2 inhibitors of the invention may be applied as monotherapy or may be one or more other antitumor agents, such as mitosis inhibitors such as vinblastine; Alkylating agents such as cis-platin, carboplatin and cyclophosphamide; Antimetabolites such as 5-fluorouracil, cytosine arabinoside and hydroxyurea, or one of the preferred antimetabolites described, for example, in EP 239362, for example N- ( 5- [N- (3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl) -N-methylamino] -2-tenoyl) -L-glutamic acid; Growth factor inhibitors; Cell cycle inhibitors; Intercalating antibiotics such as adriamycin and bleomycin; Enzymes such as interferon; And anti-hormones such as antiestrogens such as nolvadex [Nolvadex ™ (tamoxifen)] or for example antiandrogens such as cassodex [4'-cyano- 3- (4-fluorophenylsulfonyl) -2-hydroxy-2-methyl-3 '-(trifluoromethyl) propionanilide)]. Such combined treatment can be accomplished by administering the individual treatment components simultaneously, sequentially or separately.

Pharmaceutical compositions may be, for example, in forms suitable for oral administration, eg, tablets, capsules, pills, powders, sustained release formulations, solvents, suspensions; Forms suitable for parenteral injection, such as sterile solvents, suspensions or emulsions; Forms suitable for topical administration, such as ointments or creams; Or in a form suitable for rectal administration, eg suppositories. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages. Pharmaceutical compositions will comprise conventional pharmaceutical carriers or excipients and the compounds according to the invention as active ingredients. It may also include other medical or pharmaceutical agents, carriers, adjuvants and the like.

Examples of parenteral dosage forms include solvents or suspending agents of the active compounds in sterile aqueous solutions, such as aqueous propylene glycol or dextrose solutions. Such dosage forms can optionally be buffered as appropriate.

Suitable pharmaceutical carriers include inert diluents or fillers, water and various organic solvents. Pharmaceutical compositions may optionally contain additional ingredients such as flavors, binders, excipients, and the like. Thus, in the case of oral administration, tablets containing various excipients, such as citric acid, may be used for various disintegrants such as starch, alginic acid and certain complex silicates, and binders such as sucrose, gelatin and Can be used with acacia. In addition, lubricants such as magnesium stearate, sodium lauryl sulfate and talc are often useful in the tableting process. Solid compositions of a similar type may also be used in soft and hard filled gelatin capsules. Preferred materials for this include lactose or lactose and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration, the active compounds in such formulations, in combination with diluents such as water, ethanol, propylene glycol, glycerin or combinations thereof, may be used in various sweetening or flavoring agents, Colorants or dyes, and optionally, emulsifiers or suspending agents.

Methods for preparing various pharmaceutical compositions having specific amounts of active compounds are known or will be apparent to those skilled in the art. See, eg, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easter, Pa., 15th Edition (1975). )].

The examples and preparations provided below illustrate the method of the present invention. It should be appreciated that the scope of the present invention is not limited to the scope of the following examples and preparations.

Unless otherwise indicated, the "test compound" used in the following examples is a selective erbB2 inhibitor E-2-methoxy-N- (3- {4- (3-methyl-4- (6-methyl-pyridine-) 3-yloxy) -phenylamino) -quinazolin-6-yl} -allyl) -acetamide.

Example  One

FRE  Model: Effect of Duration of Exposure on Antitumor Efficacy of Test Compound

The purpose of the pre-clinical study was to determine whether the C _max or area under the curve (AUC) of the test compound is a decisive factor for antitumor efficacy. An additional goal was to establish a pharmacokinetic / pharmacodynamic (PK / PD) relationship in the FRE / erbB2 tumor model. FRE / erbB2 is an engineered murine tumor model that overexpresses human erbB2 with transmembrane mutations.

The role of the test compound exposure period on FRE / erbB2 tumor growth in athymic mice was determined. Test compounds were administered using tail vein infusion or orally. A tail vein infusion was used to maintain a fixed C _max (1200 ng / ml) concentration during the daily infusion throughout the exposure period, thus varying the AUV. Plasma concentrations and treatments in treated animals are shown in Table 1.

A 1.15 mg / ml solution of test compound was injected intravenously by gradient injection at 550 μl / hr for 2 minutes, followed by 50 μl / hr for 15 minutes or 4 hours daily. (Based on CI of test compound). Athymic female mice bearing FRE / erbB2 tumors (approximately 100 mm 3 in size) were treated with vehicle or administered orally or intravenously with test compounds. Body weight changes and tumor measurements were obtained at regular intervals (day 1, 3, 5 and 7). This study was conducted for 7 days. At the end of the study, plasma and tumor samples were separated for PK analysis and PD analysis. The results for inhibition of p-erbB2 (phosphorylated form of the erbB2 receptor) as well as antitumor efficacy, tumor volume, weight change, plasma concentration of the test compound in control and test compound animals are shown in Table 1 below:

TABLE 1

The values in parentheses are the average weight in grams; *: Compared to vehicle (IV) group.

PO, QD study N = 6; IV, QD study N = 4

% Gl =% growth inhibition.

In animals treated by daily oral administration of test compounds, approximately 54% tumor growth inhibition was achieved. Plasma concentration at 0.5 hours after dosing on day 7 was 1460 ng / ml. The test compound treatment was safe and did not cause any weight loss or mortality.

Injecting the test compound for 15 minutes daily inhibited approximately 34% tumor growth. In contrast, equal infusion for 4 hours daily resulted in substantially higher tumor growth inhibition (76%). This suggests that the coverage period above the threshold plasma concentrations has important implications for the overall antitumor efficacy of the test compound in this model. Based on these results, one may conclude that coverage for 4 hours per day (AUC) at a plasma concentration close to 500 ng / ml is sufficient to induce significant FRE / erbB2 tumor growth inhibition. The duration of exposure or the AUC (coverage) period significantly affects efficacy; _Daily C _max alone cannot explain the efficacy in this model.

The coverage period (approximately 4 hours per day) at plasma concentrations of approximately 500 ng / ml had an advantage over the shorter coverage period (approximately 15 minutes per day) in the FRE / erbB2 tumor model.

The antitumor efficacy of 25 mg / kg of test compound administered orally once daily was effective to slow the volumetric growth of FRE tumors in nu / nu mice, which is shown in the bar graph format of FIG. This figure showed that in 7 days of treatment, the FRE tumor volume of treated mice was about half of the control.

Figure 2 shows the antitumor efficacy of 10 mg / kg of test compound administered intravenously for 7 days for 4 hours daily based on an absolute baseline and injecting about 1.4 mg / kg of inhibitor or vehicle for about 15 minutes daily It is shown in bar graph format that it is highly effective in both cases when compared to one. About 10 mg / kg of test compound slowed tumor volume increase below 24% of vehicle control. In contrast, rapid infusion of about 1.4 mg / kg slowed tumor growth to less than 66% of vehicle control.

Example  2

SK - OV -3 model: effect of duration of exposure on antitumor efficacy of test compound

Pre-clinical studies were conducted to determine whether the test compound coverage period was a decisive factor for antitumor efficacy. Another goal was to establish a human ovarian adenocarcinoma, SK-OV-3 tumor model with a minimum effect (C _max and Cave _{0 -4} h) concentration in the.

As background, test compounds (PO, QD) were found to be potent against FRE erbB2 tumors in Example 1. Similarly, IV (intravenous) administration of test compounds was efficacious against FRE erbB2 tumors. These findings indicate that maintaining the blood concentration of the test compound at about 500 ng / ml for 4 hours per day, results in shorter coverage periods with the same level of p-erbB2 reduction (48-53%) in the FRE erbB2 tumor model. (About 15 minutes a day) proved advantageous. Pharmacokinetics, pharmacodynamics and efficacy data are shown in Table 1.

Than when based on the exposure measured in earlier studies, is about 1200 ng / ml of C _max, or from about 985 ng.hr/ml AUC _{0 -2} h for the test compound using a coverage of about 2 hours about 50% FRE erbB2 was crucial for inhibiting tumor growth.

The study was extended to the human ovarian adenocarcinoma model SK-OV-3, a human xenograft model that overexpresses erbB2.

SK-OV-3 cells obtained from ATCC (Rockville, MD) were grown in McCoy's medium containing 10% fetal bovine serum and pen / strep. Exponentially growing cells were harvested and female athymic mice were inoculated with SC (5 million cells per animal). Athymic mice carrying SK-OV-3 tumors (size approximately 100 mm 3) were randomly divided into seven groups as shown in Table 2. On days 1, 3, 6, 10, 13 and 18, tumor measurements and body weight changes were obtained. Tumor volume was calculated by the following equation: Tumor volume (k) = (W x W) / 2 x L (L = length and W = width). At 0.5, 1, 2, 4 and 8 hours after dosing on day 18 for PK analysis, blood samples (about 50 μl) were separated. Tumors were isolated 0.5 hours after dosing on day 18 for PD analysis by ELISA. The p-erbB2 reduction rate, tumor volume and weight change in control and test compound treated animals are shown in Table 2 below:

TABLE 2

The values in parentheses are the average weight in grams.

TABLE 3

Pharmacokinetics of Test Compounds in SK-OV-3 Tumor-bearing Mice

Values represent mean values.

*: AUC _{0 - tlast} significant difference was observed between the AUC _{0 -4} h.

Oral antitumor efficacy of test compounds (QD and BID) against human ovarian adenocarcinoma model SK-OV-3 overexpressing erbB2 was determined. Moreover, test compound administration (QD or BID) was efficacious and resulted in dose-dependent inhibition of SK-OV-3 xenografts (FIGS. 3 and 4). The test compound was well tolerated and had no weight loss or animal mortality.

Dosing QD at 50 mg / kg of test compound for 18 days was ineffective. Approximately 29% tumor growth inhibition was achieved when a total daily dose of 50 mg / kg per day was administered according to the BID schedule (25 mg / kg, BID). The decrease in erbB2 receptor autophosphorylation at 0.5 hours after dosing on day 18 was equivalent in both QD and BID treated groups (14-20%), but the C _max for test compounds in the 50 mg / kg QD group was 25 mg / kg. It was approximately 2 times higher compared to kg BID dosed animals (C _max , 3640 ng / ml vs 1780 ng / ml). Similarly, AUC _{0 -4} h (3410 ng.hr/ml about 1560 ng.hr/ml) and _{Cave 0 -4 h (853 ng /} ml vs. 390 ng / ml) in QD group BID treatment groups and the It was about two times higher in comparison. These results demonstrated that neither higher C _max or AUC _{0 -4} h was critical for the antitumor efficacy of the test compound. Twice a day (BID) The average coverage (Cave _{0 -4} hr) of 390 ng / ml of test compound is an advantage over the mean coverage (Cave _0-4 hr) of 853 ng / ml (QD) once a day Both approaches (QD & BID) provided equivalent levels of erbB2 autophosphorylation reduction.

Advantages of BID dosing over QD dosing have also been observed with higher doses of test compounds in the SK-OV-3 model. Compared to the 50 mg / kg BID dose (100 mg / kg per day) of the test compound, the QD dose of 100 mg / kg per day resulted in a higher reduction in erbB2- autophosphorylation (75% vs. 24%). Which means higher C _max (12,100 ng / ml vs. 3880 ng / ml), AUC _{0 -4} h (16,300 ng.hr/ml vs. 4180 ng.hr/ml) and Cave _{0 -4} h (4080 ng / ml vs. 1050 ng / ml). However, the QD schedule was less effective than the BID schedule (23% versus 45% tumor growth inhibition). These results indicate that the test compound high C _max or AUC _{0 -4} h the number of substantive benefit, while any that are not provide, at least coverage threshold level in the tumor model than the (frequency) (Cave _{0 -4,} BID versus QD) It is supported by the fact that is a determinant of anti-tumor efficacy. Moreover, if the average duration of the coverage is maintained for a long time with BID dosing, then approximately 24% reduction of SK-OV-3 tumor p-erbB2 may be sufficient for approximately 50% growth inhibition.

At 200 mg / kg QD dosing, the oral absorption rate of the test compound was non-linear. C _max and Cave _{0 -4} h values for the test compound was 200 mg / kg QD was equivalent from the animals and the dose 100 mg / kg BID dosing all animals. In spite of lower tumor erbB2- autophosphorylation reduction in animals administered 100 mg / kg BID (62% vs. 90%), tumor growth inhibition rate in these groups was 200 mg / kg, 2 times higher than in QD-treated animals. Higher (71% vs. 36%). These observations add to the fact that lower reductions in erbB2-autophosphorylation (62% vs. 90%) have significant advantages with longer / more frequent daily coverage (BID schedules) at equivalent C _max . Backed by.

The findings were consistent with the results in athymic mice carrying FRE erbB2 tumors (Example 1). In that study, it was advantageous to maintain the blood concentration of the test compound at about 500 ng / ml for 4 hours per day with an equivalent reduction in erbB2- autophosphorylation compared to 15 minutes per day.

Thus, what was found using the SK-OV-3 tumor model in this example is the fact that the total daily coverage, ie the number of daily doses, is crucial for the antitumor efficacy of the test compound. In other words, the BID schedule was advantageous over QD dosing. It is of limited value that the higher rate of erbB2- autophosphorylation decreases over a shorter period.

Example  3

Effect of Duration of Exposure on Antitumor Efficacy of Test Compounds

To determine whether the test compound coverage period is a decisive factor for anti-tumor efficacy, and also to establish pre-clinical (C _max and Cave _0-4 h) concentrations in human breast adenocarcinoma, BT-474 tumor model The study was conducted.

As background, test compounds (PO, QD) were found to be potent against FRE erbB2 tumors in Example 1. Similarly, IV (intravenous) administration of test compounds was efficacious against FRE erbB2 tumors. These findings indicate that maintaining the blood concentration of the test compound at about 500 ng / ml for 4 hours per day, results in a shorter coverage period with an equivalent level of p-erbB2 reduction (48-53%) in the FRE erbB2 tumor model. (About 15 minutes a day) proved advantageous. Pharmacokinetics, pharmacodynamics and efficacy data are shown in Table 1.

Based on the exposure measured in the earlier study in the FRE erbB2 model, the study was extended to the ovarian adenocarcinoma xenograft model SK-OV-3 overexpressing erbB2 in Example 2. The test compound was efficacious and what was found using the SK-OV-3 tumor model is that the total daily coverage, ie the number of doses per day, is crucial for the antitumor efficacy of the test compound. The BID dosing schedule was more advantageous than the QD dosing schedule. It is of limited value that the higher rate of erbB2- autophosphorylation decreases over a shorter period.

In this example, to determine the antitumor efficacy of the test compound against the human breast adenocarcinoma model BT-474 overexpressing the erbB2 receptor, the scope was expanded to evaluate the significance (importance) of the number of doses per day.

Exponentially growing BT-474 cells (RPM 1640 [Gibco] with 10 mM HEPES, 10% PBS and pen / strep) were harvested and female athymic mice were inoculated with SC (5 million cells per animal). . A trochar piece of BT-474 tumor was then implanted in the right flank of the animal. BT-474 tumor bearing mice (size 50-320 mm 3, N = 40) were randomly divided into seven groups of 5 to 6 animals each. Animals were treated with vehicle (PO, BID) or with test compounds (PO, QD or BID) as described in Table 4. On days 1, 6, 11, 15 and 22, tumor measurements and body weight changes were obtained. Tumor volume was calculated by the following equation: Tumor volume (k) = (W x W) / 2 x L (L = length and W = width). At 0.5, 1, 2, 4 and 8 hours after dosing on day 22 for PK analysis, blood samples (about 50 μl) were isolated. Tumors were isolated at 0.5 hours after dosing on day 22 for PD analysis by ELISA.

Statistical Analysis: AVONA was performed based on% growth data and planned comparisons were made between quasi-doses. Due to the distribution of these values, the data were log-transformed for analysis. For multiple comparison analysis, Dunnett-Tamahane procedure was used. The p-erbB2 reduction rate, tumor volume and weight change in control and test compound treated animals are shown in Table 4 below:

TABLE 4

The values in parentheses are the average weight in grams.

The pharmacokinetics of test compounds in BT-474 tumor bearing mice are shown in Table 5 below:

TABLE 5

nd: not determined due to the extrapolated portion of AUC ≧ 30% of total AUC.

Values represent mean values.

*: The values were evaluated based on the extrapolated concentration 4 hours from 2 to 8 hour exposure.

Therefore, the oral antitumor efficacy of test compounds (QD and BID) against human breast adenocarcinoma model BT-474 overexpressing erbB2 was determined. Test compound administration (QD or BID) was efficacious and caused growth inhibition of BT-474 xenografts (FIGS. 5A and 5B). The test compound was well tolerated and had no weight loss or animal mortality. Since the variation in initial tumor volume was large, the percent growth rate of individual tumors was calculated and the relative antitumor efficacy was determined using the average of each group.

Treatment of test compounds with 15 mg / kg QD (15 mg / kg per day) and BID (30 mg / kg per day) for 22 days was efficacious, with 22% and 54% (p = 0.007) tumor growth inhibition rates Were each induced. At 0.5 hours after dosing on day 22, the rate of reduction of erbB2 receptor autophosphorylation was below the detection limit in both QD and BID treated groups, and measurement of Cave _{0 -4} h in QD dosed animals was extrapolated by AUC ≥30% of total AUC. It was not possible due to the part. 15 mg / kg, BID efficacy C _max, AUC _{0 -4} h and Cave _{0 -4} h (54% growth inhibition) against a test compound from a dosage animal is 616 ng / ml, 480 ng.hr/ml, respectively, and 120 ng / ml.

After treatment with 30 mg / kg QD (30 mg / kg per day) and BID (60 mg / kg per day), PK, PD and anti-tumor efficacy of the test compounds were also determined. PK value was equivalent to the test compound after QD or BID dosing day 22 the measured, i.e., C _max (1800 ng / ml vs. _{1570 ng / ml), AUC 0} -4 h (1280 ng.hr/ml vs. 1440 ng .hr / ml) and was _{Cave 0 -4 h (320 ng /} ml vs. 360 ng / ml, Table 5). BT-474 tumor erbB2 autophosphorylation reduction in QD dosed animals was higher than in BID dosed animals (57% vs. 26%, p = 0.06). The 30 mg / kg BID schedule of the test compound was more potent than the QD dosing schedule (68% vs. 33% growth inhibition, p = 0.053).

Compared to the test compound dosed at 30 mg / kg QD or BID (30 mg / kg per day or 60 mg / kg per day), 50 mg / kg per day is administered with QD or BID (50 mg / kg per day or At 100 mg / kg per day, tumor erbB2- autophosphorylation decreased (approximately 75% reduction). PK parameters of the test compounds at 22 days after 50 mg / kg QD or BID treatment groups was also the same level, that is, C _max (5890 ng / ml vs. _{6170 ng / ml), AUC 0} -4 h (4220 ng.hr/ ml was for 5280 ng.hr/ml) and _{Cave 0 -4 h (1060 ng /} ml vs. 1320 ng / ml). QD schedules were considered to be less potent than BID schedules (35% vs. 68% tumor growth inhibition, p = 0.066).

An integrated test was performed to compare quasi-doses between QD and BID. The results revealed that overall BID dosing was more effective than QD dosing (p = 0.0346). This finding suggested that the test compound-dose multiplicity had a positive effect on the overall treatment (treatment) results.

Of the test compounds observed in the 50 mg / kg, QD (50 mg / kg daily) vs. 30 mg / kg, BID (60 mg / kg daily) groups (the two closest in total daily dosing schedule) PK, PD and anti-tumor efficacy were compared and evaluated to determine the value of dose-count. The p-erbB2 reduction in the 50 mg / kg, QD (50 mg / kg daily) group was significantly higher than in the 30 mg / kg, BID (60 mg / kg daily) group (75% vs. 26%). p-erbB2 reduction rate, Table 4). Similarly, 30 mg / kg, compared to the BID dose group, and high C _max than for the test compound (5890 ng / ml vs. _{1570 ng / ml), AUC 0} -4 h (4220 ng.hr/ml vs. 1440 ng was observed in .hr / ml) and _{Cave 0 -4 h (1060 ng /} ml vs. 360 ng / ml) a 50 mg / kg, QD dosing groups (Table 5). Lower p-erbB2 reduction and PK- values than for the test compound (that is, C _max, AUC _{0 -4} h and Cave _{0 -4} h), and even, 30 mg / kg, BID dosing (60 mg / kg 1 il though ) Was more effective than 50 mg / kg, QD dosing (50 mg / kg per day). Overall, approximately 68% and 35% tumor growth inhibition was observed in the 30 mg / kg, BID and 50 mg / kg, QD groups, respectively (p = 0.0636). Although the total daily doses of test compounds in these two groups are slightly the same, it can be concluded that the number of daily doses, ie, BID doses, is advantageous over QD doses.

These results suggest that the in that imparts the advantage, as compared to the number of daily dosage, i.e., coverage twice a day coverage Cave _0-4 is once a day involving a QD dosage involving BID dosing Cave _{0 -4} Similar to the results according to the SK-OV-3 tumor model study of Example 2. Moreover, if the average duration of said coverage (about 360 ng / ml) is maintained for a long time with BID dosing, then for approximately 50% growth inhibition, BT-474 tumor-autophosphorylation of BT-474 tumors twice daily with BID dosing Approximately a 26% reduction may be sufficient. These findings are consistent with the results of intravenous administration by injecting test compounds into FRE erbB2 tumor bearing athymic mice. The study demonstrated that maintaining the blood concentration of the test compound at about 500 ng / ml for 4 hours per day provided an advantage over bolus administration.

Thus, the findings from the BT-474 tumor model suggest that both dosing multiplicity and daily dosing are critical factors for antitumor efficacy of test compounds. Dosage multiplicity relates to the administration of the same dose (X mg / kg) twice or more to six times per day or optionally seven times per day compared to administration of a constant dose (X mg / kg) once a day. will be. The number of doses per day relates to dividing the daily dose into several doses, e.g. a 1/2 X mg / kg dose twice daily compared to an X mg / kg dose once daily. Relates to administration.

It is of limited value that the higher rate of erbB2- autophosphorylation decreases over a shorter period.

Example  4

Effect of Duration of Exposure on Antitumor Efficacy of Test Compounds

To determine whether the test compound coverage period is a decisive factor for antitumor efficacy, and also to establish at least potent (C _max and Cave _0-4 h) concentrations in the human breast adenocarcinoma tumor model MDA-MB-453. Clinical studies were performed.

As background, test compounds (PO, QD) were found to be potent against FRE erbB2 tumors in Example 1. Similarly, IV (intravenous) administration of test compounds was efficacious against FRE erbB2 tumors. These findings suggest that maintaining a blood concentration of the test compound at about 500 ng / ml for 4 hours per day, results in shorter coverage periods with an equivalent level of p-erbB2 reduction (48-53%) in the FRE erbB2 tumor model. (About 15 minutes a day) proved advantageous. Pharmacokinetics, pharmacodynamics and efficacy data are shown in Table 1.

The study was extended to SK-OV-3, a human ovarian adenocarcinoma xenograft model that overexpresses erbB2. The test compound was efficacious and the findings using the SK-OV-3 tumor model indicate that the total daily coverage, i.e. the number of doses per day, is crucial for the antitumor efficacy of the test compound (BID dosing schedule is QD More favorable than dosing schedule). The antitumor effect of the QD versus BID oral dosing schedule of test compounds against the BT-474 human breast adenocarcinoma model overexpressing erbB2 was also studied. The findings from this also suggested that dosing multiplicity and dosing frequency were critical to the antitumor efficacy of the test compound. Overall, the findings using the SK-OV-3 and BT-474 models were of limited value in the higher rate of erbB2- autophosphorylation reduction over a shorter period.

This study was performed to determine the oral antitumor efficacy of test compounds against an additional human breast carcinoma model MDA-MB-453 overexpressing erbB2. The second purpose of this study was to determine what benefits the test compound dosing multiplicity or dosing frequency had over this model.

Study plan: harvesting exponentially growing MDA-MB-453 cells (DMEM / F12 [Gibco] with 10% PBS and pen / strep) and collecting SC (5 million cells per animal) in female athymic mice Inoculation. MDA-MB-453 tumor bearing mice (size approximately 100 mm 3, N = 64) were randomly divided into 8 groups of 8 animals each. Animals were treated with vehicle (PO, QD or BID) or with test compounds (PO, QD or BID) as described in Table 6. On days 1, 3, 7, 10, 14, 17, 21, 24 and 29, tumor measurements and body weight changes were obtained. Tumor volume was calculated by the following equation: Tumor volume (k) = (W x W) / 2 x L (L = length and W = width). At 0.5, 1, 2, 4 and 8 hours after dosing on day 29 for PK analysis, blood samples (about 50 μl) were separated. Tumors were isolated 0.5 hours after dosing on day 29 for PD analysis by ELISA.

Statistical Analysis: AVONA was performed based on% growth data and planned comparisons were made between quasi-doses. Due to the distribution of these values, the data were log-transformed for analysis. For multiple comparison analysis, the Dunnett-Tamahan procedure was used.

The p-erbB2 reduction rate, tumor volume and weight change in control and test compound treated animals are shown in Table 6 below:

TABLE 6

The values in parentheses are the average weight in grams.

The pharmacokinetics of test compounds in MDA-MB-453 tumor bearing mice are shown in Table 7 below.

TABLE 7

Values represent mean values.

Therefore, the oral antitumor efficacy of test compounds (QD and BID) against human breast adenocarcinoma model MDA-MB-453 overexpressing erbB2 was determined. Test compound administration (QD or BID) was efficacious and caused growth inhibition of MDA-MB-453 xenografts (FIGS. 6A and 6B). The test compound was well tolerated and had no weight loss or animal mortality.

Treatment of test compounds with 50, 100 and 200 mg / kg QD (50, 100 and 200 mg / kg per day) for 29 days was efficacious, causing 38%, 63% and 100% tumor growth inhibition, respectively. I was. In the 50, 100 and 200 mg / kg groups, the reduction rates of erbB2 receptor autophosphorylation were 78%, 88% and 92% at 0.5 hours after dosing on 29 days. BID dosing of test compounds at 25, 50 and 100 mg / kg for 29 days was efficacious against MDA-MB-453 tumors and resulted in 19%, 66% and 83% growth inhibition rates, respectively. The p-erbB2 reduction rates in these groups were 69%, 75% and 79%, respectively.

ANOVA was used to statistically analyze the overall efficacy of different doses of test compounds. Dunnett-Tamahan's procedure was used to make multiple comparisons with vehicle adjustments. As a result, 25 mg / kg BID and 50 mg / kg QD (p = 0.295), 50 mg / kg BID and 100 mg / kg QD (p = 0.703), and 100 mg / kg BID and 200 mg / No significant differences (significant differences) were found between the kg QD (p = 0.117) dosing schedules. Similarly, there was no significant difference between similar doses, ie 50 mg / kg BID vs. 50 mg / kg QD (p = 0.13), and 100 mg / kg BID vs. 100 mg / kg QD (p = 0.17). Comparing statistical assessments using only antitumor efficacy and dose / dose schedules observed in different groups leads to a final conclusion that focuses on the task, namely, how the BID schedules of test compounds have advantages over QD dosing. It was not enough.

The reduction rate of p-erbB2 after dosing QD (50-200 mg / kg) or BID (25-100 mg / kg) was 69-92%, which was difficult to use as a parameter for further statistical data analysis. Thus, the pharmacokinetic parameter of the test compound, that is, by using the C _max and Cave _{0 -4} h expanded data analysis.

50 mg / kg (1 day 50mg / kg) and 100 mg / kg of a 591 ng / ml and 3120 ng / ml to give (1 day 100 mg / kg) after QD dosing Cave _{0 -4} h is 38% and 63% Tumor growth inhibition was induced. 50 mg / kg BID dosing schedule in accordance with one days _{0 -4} 2 Cave of 509 ng / ml obtained twice h is brought to 66% efficacy. Of 509 ng / ml was maintained for 8 hours per day in accordance with the BID dosing Cave _{0 -4} h is 591 ng / ml (50 mg / kg QD dosing) for 4 hours a day or 3120 ng / ml (100 mg / kg QD dosage) (there was no significant difference as to keep the Cave _{0 -4} h of p = 0.13 & p = 0.58), respectively. This can be interpreted as maintaining an average plasma concentration of 509 ng / ml for 8 hours per day compared to maintaining an average plasma concentration of 591 to 3120 ng / ml for 4 hours per day. C _max for the test compound in the 50 mg / kg QD group and the 50 mg / kg BID group was equivalent (comparative) (2760 ng / ml vs. 2390 ng / ml), whereas C in the 100 mg / kg QD group _max was approximately 4 times higher (9770 ng / ml). These results suggest that when the rate of p-erbB2 reduction is equal, higher C _max or Cave _{0 -4} h alone have limited value.

To 100 mg / kg BID and 200 mg / kg QD group compared to the C _max and Cave _{0 -4} h on anti-tumor efficacy of the test compound observed in the addition was carried out. C _max for the test compound in the 200 mg / kg QD group was about 2.4 times higher than C _max for the test compound in the 100 mg / kg BID group (16700 ng / ml vs. 6870 ng / ml). Similarly, Cave _{0 -4} h is about 3.8 times higher in the 200 mg / kg QD group compared to the 100 mg / kg BID group (6510 ng / ml vs. 1710 ng / ml). Despite the higher C _max and Cave _{0 -4} h, the overall efficacy of the test compound observed with the 200 mg / kg QD dose was comparable to the antitumor efficacy observed with the 100 mg / kg BID dose ( 100% vs 83%). These data indicate that maintaining a mean plasma concentration of 1710 ng / ml (C _max , 6870 ng / ml) for 8 hours per day by administering 100 mg / kg BID of the test compound is 6510 ng / ml after 200 mg / kg QD dosing ( C _max , 16,700 ng / ml), suggesting that efficacy was at the same level as maintaining average plasma concentrations.

Thus, these findings indicate that in the MDA-MB-453 tumor model, maintaining the plasma concentration of the test compound at about 509 ng / ml for 8 hours per day (50 mg / kg, BID dosing) in inhibiting tumor growth. It was shown to be effective at the same level as maintaining an average plasma concentration of 591 to 3120 ng / ml (50-100 mg / kg QD dose) for 4 hours per day. Thus, low doses of test compounds given according to the BID schedule were efficacious at equivalent levels to higher doses provided according to the QD schedule.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention other than those described herein will be apparent to those skilled in the art from the foregoing specification and the accompanying drawings. Such modifications fall within the scope of the appended claims.

All patents, patent applications, publications, test methods, literature and other materials cited herein are hereby incorporated by reference in their entirety.

The present invention provides an improved dosing schedule for anticancer agents.

Claims (1)

(a) administering to a mammal in need thereof a therapeutically effective amount of a first inhibitor of the erbB2 receptor; And (b) subsequent administration of said therapeutically effective amount of a second inhibitor of the erbB2 receptor in one to six doses to said mammal after a certain interval comprising less than 24 hours. Comprising, the overexpression of an erbB2 receptor in said mammal.

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